Tetrapartate prodrugs

ABSTRACT

A compound of Formula I, providing tetrapartate prodrugs is provided  
                 
 
     wherein:  
     L 1  is a bifunctional linking moiety;  
     D is a moiety that is a leaving group, or a residue of a compound to be delivered into a cell;  
     Z is covalently linked to [D] y , wherein Z is selected from the group consisting of:  
     a moiety that is actively transported into a target cell, a hydrophobic moiety, and combinations thereof,  
     Y 1 , Y 2 , Y 3  and Y 4  are each independently O, S, or NR 12 ;  
     R 11  is a mono- or divalent polymer residue;  
     R 1 , R 4 , R 9 , R 10  and R 12  are independently selected from the group consisting of hydrogen, C 1-6  alkyls, C 3-12  branched alkyls, C 3-8  cycloalkyls, C 1-6  substituted alkyls, C 3-8  substituted cycloalkyls, aryls, substituted aryls, aralkyls, C 1-6  heteroalkyls and substituted C 1-6  heteroalkyls;  
     R 2 , R 3 , R 5  and R 6  are independently selected from the group consisting of hydrogen, C 1-6  alkyls, C 1-6  alkoxy, phenoxy, C 1-8  heteroalkyls, C 1-8  heteroalkoxy, substituted C 1-6  alkyls, C 3-8  cycloalkyls, C 3-8  substituted cycloalkyls, aryls, substituted aryls, aralkyls, halo-, nitro- and cyano-, carboxy-, C 1-6  carboxyalkyls and C 1-6  alkylcarbonyls;  
     Ar is a moiety which when included in Formula (I) forms a multi-substituted aromatic hydrocarbon or a multi-substituted heterocyclic group;  
     (m), (r), (s), (t), and (u) are independently zero or one;  
     (p) is zero or a positive integer; and (y) is 1 or 2;  
     together with methods of preparing and using these new tetrapartate prodrugs.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pending U.S.patent application Ser. No. 09/183,557, filed on Oct. 30, 1998, which inturn is a continuation-in-part of U.S. patent application Ser. No.08/992,435, filed on Dec. 17, 1997, now abandoned, the contents of bothof which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to tetrapartate prodrugs. Inparticular, the invention relates to polymer conjugates that providetetrapartate prodrugs that deliver active agents linked to uptakeenhancing moieties effective to provide enhanced efficacy, e.g., asantitumor agents or the like.

BACKGROUND OF THE INVENTION

[0003] Over the years, several methods of administeringbiologically-effective materials to mammals have been proposed. Manybiologically-effective materials, e.g., including medicinal agents andthe like, are available as water-soluble salts and can be included inpharmaceutical formulations relatively easily. Problems arise when thedesired biologically-effective material is either insoluble in aqueousfluids or is rapidly degraded in vivo. For example, alkaloids are oftenespecially difficult to solubilize.

[0004] One way to solubilize biologically-effective material(s) is toinclude them as part of a soluble prodrug. Thus, prodrugs includechemical derivatives of a biologically-active material, or parentcompound which, upon administration, eventually liberate the parentcompound in vivo. Prodrugs allow the artisan to modify the onset and/orduration of action of an agent in vivo and can modify thetransportation, distribution or solubility of a drug in the body.Furthermore, prodrug formulations often reduce the toxicity and/orotherwise overcome difficulties encountered when administeringpharmaceutical preparations.

[0005] Typical examples of prodrugs include organic phosphates or estersof alcohols or thioalcohols. See Remington's Pharmaceutical Sciences,16th Ed., A. Osol, Ed. (1980), the disclosure of which is incorporatedby reference herein.

[0006] Prodrugs are, by definition, forms of the parent or activecompound. The rate of release of the active drug, typically, but notexclusively, by hydrolysis of the prodrug, is influenced by severalfactors, but especially by the type of bond joining the active drug tothe modifier. Care must be taken to avoid preparing prodrugs which areeliminated through the kidney or reticular endothelial system, etc.,before a sufficient amount of the parent compound is released. Byincorporating a polymer as part of the prodrug system, one can increasethe circulating half-life of the drug. However, in some situations, suchas with alkaloids, it has been determined that when only one or twopolymers of less than about 10,000 daltons are conjugated thereto, theresulting conjugates are rapidly eliminated in vivo especially if asomewhat hydrolysis-resistant linkage is used. In fact, such conjugatesare so rapidly cleared from the body that even if a hydrolysis-proneester linkage is used, not enough of the parent molecule is regeneratedin vivo. This is often not a concern with moieties such as proteins,enzymes and the like, even when hydrolysis-resistant linkages are used.In those cases multiple polymer strands, each having a molecular weightof about 2-5 kDa, are used to further increase the molecular weight andcirculating half-life.

[0007] One way in which these problems have been addressed is described,for example, by co-owned patent applications Ser. No. 09/183,557, filedOct. 30, 1998 and Ser. No. 08/992,435, filed on Dec. 17, 1997. Theseteach double prodrugs, i.e., tripartate, that comprise polymerconjugates of various biologically-effective materials, and methods ofmaking these conjugates. The double prodrug linkages are selected tohydrolyze in vivo at a rate which generates sufficient amounts of the“second” and more reactive prodrug compound within a suitable time afteradministration by, e.g., a 1,4-aryl or 1,6-aryl (e.g., benzyl)elimination reaction, providing improved control of the pharmacokineticsof a number of small molecule drugs, agents and the like. However,further opportunities for particularly selective targeting of diagnosticand/or therapeutic agents to tissues or cells of interest, by means of arationally designed prodrug conjugate remain.

[0008] One particularly desirable target tissue for prodrugs is tumortissue. It is well known that tumors generally exhibit abnormal vascularpermeability characterized by enhanced permeability and retention (“EPReffect”). This EPR effect advantageously allows biologically-effectivematerials, in the form of macromolecules, e.g., protein(s) such asenzymes and/or antibodies and derivatives or fragments thereof, or thelike, to readily enter tumor interstitial tissue space (see, forexample, the review article by Maeda et al., 2000, J. of ControlledRelease, 65:271-284, incorporated by reference herein). Certain othertissues, in addition to tumors, can exhibit this same EPR effect, underconditions of inflammation, and the like.

[0009] In brief, and without being bound by any theory or hypothesis asto the working of the EPR effect, it is believed that the EPR effectallows penetration of large molecule or macromolecule substances,including polymer-based delivery systems. This provides a substantiallyselective delivery of polymer conjugates into tumor tissue space, e.g.,tumor interstitial space. Thereafter, however, the same EPR effect isbelieved to allow the released prodrug(s) and/or any newly releasedrelatively low molecular weight, biologically-effective materials, torapidly diffuse out of the extracellular tissue space of the targetedtissue. It is believed that if the released active agent fails to betaken up by the surrounding cells at a sufficient rate, they diffuseaway from the release site in the ongoing blood or lymphatic flow.

[0010] Thus, there continues to be a need to provide additionaltechnologies for forming prodrugs which would benefit from the multiplelevel prodrug concept and compensate or control for the EPR effect byallowing for more rapid update or transport of the releasedbiologically-effective materials into tumor cells and/or cells of othertissues of interest that exhibit the EPR effect.

SUMMARY OF THE INVENTION

[0011] Broadly, the invention provides for a tetrapartate prodrug in theform of a compound of Formula I:

[0012] wherein:

[0013] L₁ is a bifunctional linking moiety.

[0014] Broadly, D is a moiety that is a leaving group, or a residue of acompound to be delivered into a cell. More particularly, D is a residueof an active biological material, or H and (y) is a positive integerequal to 1 or greater. Preferably, (y) ranges from 1 to about 5. When(y) is greater than 1, each D moiety is independently selected.

[0015] D can be any biologically active material that it is desired todeliver into a target cell or cells of an animal in need of suchtreatment, including anti-inflammatory agents, detoxifying agents,anticancer agents, and diagnostics for any of these or other conditions.

[0016] Preferably, D is an anticancer agent, an anticancer prodrug, adetectable tag, and combinations thereof. Any anticancer agent orsuitable tag that can be linked to the tetrapartate prodrug iscontemplated. Simply by way of example, these include an anthracyclinecompound, a topoisomerase I inhibitor, daunorubicin, doxorubicin;p-aminoaniline to name but a few.

[0017] When D is a leaving group, D can be, e.g.,N-hydroxybenzotriazolyl, halogen, N-hydroxyphthalimidyl, p-nitrophenoxy,imidazolyl, N-hydroxysuccinimidyl and/or thiazolidinyl thione.

[0018] Z is covalently linked to [D]_(y), wherein Z is a moiety that isactively transported into a target cell, a hydrophobic moiety, andcombinations thereof. Optionally, Z is monovalent, multivalent, or morepreferably, bivalent, wherein (y) is 1 or 2. Z itself optionallyincludes an amino acid residue, a sugar residue, a fatty acid residue, apeptide residue, a C₆₋₁₈ alkyl, a substituted aryl, a heteroaryl,—C(═O), —C(═S), and —C(═NR₁₆), where are R₁₆ is as defined below.

[0019] When Z includes at least one amino acid residue, the amino acidis, e.g., alanine, valine, leucine, isoleucine, glycine, serine,threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan,aspartic acid, glutamic acid, lysine, arginine, histidine, proline,and/or a combination thereof, to name but a few. When Z includes apeptide, the peptide ranges in size, for instance, from about 2 to about10 amino acid residues. In one preferred embodiment, the peptide isGly-Phe-Leu-Gly or Gly-Phe-Leu.

[0020] In addition, Y₁ through Y₄ are independently O, S, or NR₁₂; andR₁₁ is a mono- or divalent polymer residue.

[0021] R₁, R₄, R₉, R₁₀, R₁₂ and R₁₆ are independently hydrogen, C₁₋₆alkyl, C₃₋₁₂ branched alkyl, C₃₋₈ cycloalkyl, C₁₋₆ substituted alkyl,C₃₋₈ substituted cycloalkyl, aryl, substituted aryl, aralkyl, C₁₋₆heteroalkyl, and/or substituted C₁₋₆ heteroalkyl.

[0022] R₂, R₃, R₅ and R₆ are independently hydrogen, C₁₋₆ alkyl, C₁₋₆alkoxy, phenoxy, C₁₋₈ heteroalkyl, C₁₋₈ heteroalkoxy, substituted C₁₋₆alkyl, C₃₋₈ cycloalkyl, C₃₋₈ substituted cycloalkyl, aryl, substitutedaryl, aralkyls, halo-, nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyland/or substituted C₁₋₆ alkylcarbonyl.

[0023] Ar is a moiety which, when included in Formula (I), forms amulti-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group; wherein (m), (r), (s), (t), and (u) areindependently zero or one, (p) is zero or a positive integer. In certainpreferred embodiments, (p) is 1.

[0024] L₁ is independently one of the following:

[0025] wherein:

[0026] M is X or Q; where X is an electron withdrawing group;

[0027] Q is a moiety containing a free electron pair positioned three tosix atoms from

[0028] Simply by way of example, Q is one of the following: C₂₋₄ alkyls,cycloalkyls, aryls, and aralkyl groups substituted with a member of thegroup consisting of NH, O, S, —CH₂—C(O)—N(H)—, and/or ortho-substitutedphenyls;

[0029] X is, for instance any one of O, NR₂₀,

[0030] S, SO and SO₂;

[0031] (a) and (n) are independently zero or a positive integer; (b) iszero or one; (g) is a positive integer of 1 or greater;

[0032] (q) is three or four;

[0033] R₇, R₈, R₁₄, R₁₅, R₁₇, R₁₈ and R₂₀ are independently selectedfrom the same group as that which defines R₁; and Y₅ and Y₆ areindependently O, S, or NR₁₂. It will be appreciated that when (y) isgreater than 1, each of the D moieties are the same or different,respectively.

[0034] Preferably, (g) ranges from 1 to about 20, or more, but moretypically ranges from 1 to about 10.

[0035] Optionally,

[0036] comprises an amino acid residue, either naturally occurring ornon-naturally occurring.

[0037] R₁₁ is a mono- or bivalent polymer, e.g., having a number averagemolecular weight of from about 2,000 to about 100,000 daltons.

[0038] Methods of preparing the tetrapartate prodrugs of the inventionare also provided. In one embodiment, the method includes reacting acompound of formula (III):

[0039] with a compound of the formula (IV):

Lx—Z—[D]_(y);  (IV)

[0040] wherein B is a leaving group for Formula (III) and is defined asabove for when D is a leaving group.

[0041] Lx is a leaving group for Formula (IV) and is defined as abovefor when D is a leaving group.

[0042] L₁, Ar, Z, D, R₁-R₆, R₉-R₁₁, Y₁-Y₃, and integers are defined asabove. The reaction between (III) and (IV) is preferably conducted inthe presence of a solvent and a base. The solvent is, for example,chloroform, methylene chloride, toluene, dimethylformamide and/orcombinations thereof Dimethylformamide is generally preferred. The baseis, for example, dimethylaminopyridine, diisopropylethylamine, pyridine,triethylamine and/or combinations thereof.

[0043] Yet another method of preparing a tetrapartate prodrug of theinvention is conducted by reacting a compound of formula (V)

[0044] with a biologically active material; wherein

[0045] La is a leaving group as defined when D is a leaving group.

[0046] L₁, Ar, Z, D, R₁-R₆, R₉-R₁₁, Y₁-Y₃, and integers are defined asabove.

[0047] The reaction between (V) and the biologically active material isconducted in the presence of a coupling agent, e.g.,1,3-diisopropylcarbodiimide, a dialkyl carbodimides,2-halo-1-alkyl-pyridinium halide, 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide, 1-propanephosphonic acid cyclic anhydride, phenyldichlorophosphates, and/or combinations thereof. The reaction between(V) and the biologically active material is conducted in the presence ofa solvent and a base, e.g., as described above for the previoussynthetic method.

[0048] Methods of using the inventive tetrapartate prodrugs are alsoprovided, e.g., by treating a disease or disorder in an animal, byadministering a pharmaceutically acceptable composition comprising aneffective amount of a compound of Formula I, to an animal in needthereof. In particular, a method is provided of delivering abiologically active material D, into a cell in need of treatmenttherewith, by a process of:

[0049] administering a compound of Formula I to an animal wherein thecell is present, and wherein Formula I is hydrolyzed in vivoextracellularly to yield:

[0050] wherein Y* is the remainder of Y₂, and is independently selectedfrom the group consisting of HO—, HS—, or HNR₁₂—;

[0051] and Formula I-(i) then spontaneously hydrolyzes to

[0052] Formula I-(ii)

[0053] and CO₂,

[0054] and Formula I-(iii) Z—[D]_(y);

[0055] wherein Y** is the remainder of Y*, and is independently selectedfrom the group consisting of O, S, or NR₁₂ and

[0056] Z—[D]_(y) crosses the membrane of the cell, and is hydrolyzedtherein to release D.

BRIEF DESCRIPTION OF THE DRAWINGS

[0057]FIG. 1: Illustrates a summary of the reaction scheme for thepreparation of the tetrapartate prodrugs of doxorubicin by the methodsof Examples 1-5, wherein the aromatic is a benzyl derivative.

[0058]FIG. 2A: Illustrates a general reaction scheme for the degradationof the tetrapartate prodrug prodrug by sequential hydrolysis of thecompound of Formula I. Thus, variables of FIG. 1 are defined as forFormula I, supra. Symbols for reaction steps: (a): controllable rate invivo cleavage; (b) “first” prodrug; (c) fast reaction in presence ofwater; (d) Z—D is the “second” prodrug and it is released substantiallyinto extracellular space; (e) uptake of Z—D into cells and intracellularenzymatic hydrolysis releases D.

[0059]FIG. 2B: Illustrates a specific reaction schemes for thedegradation of tetrapartate prodrug compound identified herein ascompounds 14 (top scheme) and 17 (bottom scheme) wherein both schemesresult in a final product of doxorubicin substantially released within acell. Symbols for reaction steps are analogous to those of FIG. 2A, sothat (a), (b), (c), (d) and (e) are the analogous reaction steps for thein vivo degradation of compound 14, and that (a′), (b′), (c′), (d′) and(e′) are the analogous reaction steps for the in vivo degradation ofcompound 17. The products of step (e′) are D and Z, but the figureillustrates that much of Z further degrades into a C₁₂ acid and leucine.

[0060]FIG. 3: Illustrates a schematic of the synthesis of compound 2 asdescribed by Method 1 of Example 1.

[0061]FIG. 4: Illustrates the synthesis of compound 2, as described byMethod 2 of Example 1.

[0062]FIG. 5: Illustrates the synthesis of compound 6, as described byExample 2.

[0063]FIG. 6: Illustrates a schematic of the synthesis of compound 8, asdescribed by Example 3.

[0064]FIG. 7A: Illustrates a schematic of the synthesis of compound 10,as described by Example 4.

[0065]FIG. 7B: Illustrates a schematic of the synthesis of compound 12,as described by Example 5.

[0066]FIG. 8: Illustrates a schematic of the synthesis of compound 14,as described by Example 6.

[0067]FIG. 9: Illustrates a schematic of the synthesis of compound 17,as described by Example 7.

DETAILED DESCRIPTION OF THE INVENTION

[0068] Accordingly, the invention provides triple prodrugs compositions,hereinafter, “tetrapartate” prodrugs for delivering a biologicallyactive material into certain important target cells, such as, forinstance, tumor cells, as well as methods of making and using the same.The tetrapartate prodrug compositions of the present invention containhydrolyzable linkages between the polymer portion and a biologicallyactive material. The biologically active material is, for example, amoiety derived from a biologically active nucleophile, i.e., a native orunmodified drug or diagnostic tag. These linkages are preferably esterand/or amide linkages designed to hydrolyze at a rate which generatessufficient amounts of the biologically active parent compound in asuitable time. The term “sufficient amounts” for purposes of the presentinvention shall mean an amount which achieves a therapeutic effect.

[0069] The present invention is broadly based upon the principle thatbiologically active materials suitable for incorporation into thepolymer-based prodrug conjugates, e.g., the double prodrug compositionsas discussed supra, may themselves be substances/compounds which are notactive after hydrolytic release from the linked composition, but whichwill become active after undergoing a further chemical process/reaction,thus providing triple-acting prodrugs. These triple acting prodrugs arereferred to herein as “tetrapartate” prodrugs because the inventiveconjugates are provided in essentially four parts.

[0070] With the tetrapartate prodrugs of the invention, a therapeutic ordiagnostic agent that is delivered to the bloodstream by theabove-described double prodrug transport system) will remain inactiveuntil entering or being actively transported into a target cell ofinterest, whereupon it is activated by intracellular chemistry, e.g., byan enzyme or enzyme system present in that tissue or cell.

[0071] In particular, it has now been discovered that when certain typesof additional moieties are linked to the biologically active material aspart of the above described double prodrug conjugates, the effectivenessof many such biologically active materials is markedly increased,relative to the effectiveness seen with analogous prodrugs that lacksuch additional moiety. The tetrapartate prodrug conjugates of theinvention are thought to provide enhanced effectiveness, e.g., fortherapeutic and/or diagnostic activity, in the delivery and activity ofcertain biologically active materials, e.g., particularly small moleculetherapeutic and diagnostic agents. The tetrapartate prodrugs of theinvention prepared so that in vivo hydrolysis of the polymer-basedconjugate cleaves the conjugate so as to release the active biologicalmaterial into extracellular fluid, while still linked to the additionalmoiety. The biologically active materials are preferably, but notexclusively, small molecule therapeutic and/or diagnostic agents. Asexemplified below, in one preferred embodiment these are small moleculeanticancer agents, and the tissue to be treated is tumor tissue.

[0072] Without intending to be bound by any theory or hypothesis as tohow the invention might operate, it is believed that, depending upon theadditional moiety selected as a transport enhancer, the rate oftransport of a biologically active material into tumor cells is by thedelivery of a biologically active material into extracellular tissuespace, e.g., of a tissue exhibiting an EPR effect, in a protected and/ortransport-enhanced form. For convenience in description, the “additionalmoiety(s)” as mentioned are described herein as, “transport enhancers.”

[0073] However, in providing this convenient descriptive term, it is notintended to limit the scope of the invention solely to added moietiesthat solely enhance transport of biologically active materials intotargeted cells, since it is believed that additional or alternativemechanisms, such as protection of the Z—[D]_(y) from extracellularhydrolytic enzyme activity, may contribute to the advantages of theinventive tetrapartate prodrug.

[0074] In a further still option, the transport enhancer is selectedfrom among known substrates for a cell membrane transport system. Simplyby way of example, cells are known to actively transport certainnutrients and endocrine factors, and the like, and such nutrients, oranalogs thereof, are readily employed to enhance active transport of abiologically effective material into target cells. Examples of thesenutrients include amino acid residues, peptides, e.g., short peptidesranging in size from about 2 to about 10 residues or more, simple sugarsand fatty acids, endocrine factors, and the like.

[0075] Desirable amino acid residues include all of the knownnaturally-occurring L-amino acids. For example, L-isoleucine as atransport enhancer is exemplified in the Examples provided below.Surprisingly, it has also been discovered that D-amino acids are usefulas transport enhancers, e.g., both D and L-alanine, and other analogousamino acid optical isomers, show the same activity. Derivatives andanalogs of the naturally occurring amino acids, as well as variousart-known non-naturally occurring amino acids (D or L), hydrophobic ornon-hydrophobic, are also contemplated to be within the scope of theinvention. Simply by way of example, amino acid analogs and derivatesinclude: 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid,2,4-diaminobutyric acid, desmosine, 2,2-diaminopimelic acid,2,3-diaminopropionic acid, n-ethylglycine, N-ethylasparagine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylglycine, sarcosine, N-methylisoleucine, 6-N-methyllysine,N-methylvaline, norvaline, norleucine, ornithine, and others toonumerous to mention, that are listed in 63 Fed. Reg., 29620, 29622,incorporated by reference herein.

[0076] Short peptides are, for example, peptides ranging from 2 to about10, or more, amino acid residues, as mentioned supra. In this embodimentof the invention, it is believed that such peptide transport enhancersneed not be hydrophobic, but are thought to function in other ways toenhance uptake and/or to protect the linked small molecule agents frompremature hydrolysis in the general bloodstream. For instance, peptidetransport enhancers, and other transport enhancers of similar molecularweight ranges, are thought to sterically hinder cleavage from thebiologically active agent by plasma-based hydrolytic enzymes, but arethen cleaved within a target cell by various peptides and/or proteases,such as cathepsins.

[0077] Preferably, the transport enhancer is a hydrophobic moiety.Without meaning to be bound to any theory or hypothesis as to howhydrophobicity contributes to efficacy, it is believed that ahydrophobic moiety inhibits the extracellular cleavage of the transportenhancer away from the active biological agent, by inhibiting the attackof hydrolytic enzymes, etc. present in the extracellular tissue space,e.g., in the plasma. Thus, preferred transport enhancers include, e.g.,hydrophobic amino acids such as alanine, valine, leucine, isoleucine,methionine, proline, phenylalanine, tyrosine, and tryptophane, as wellas non-naturally occurring derivatives and analogs thereof, as mentionedsupra.

[0078] In a further option, the transport enhancer is a hydrophobicorganic moiety. Simply by way of example, the organic moiety is a C₆₋₁₈,or larger, alkyl, aryl or heteroaryl-substituted or nonsubstituted. Theorganic moiety transport enhancer is also contemplated to encompass andinclude organic functional groups including, e.g., —C(═S) and/or—C(═Y₃).

[0079] In order to appreciate the nature of the invention, severaldefinitions and explanations are provided as follows. The term,“tetrapartate” refers to prodrug conjugates, and in particular, toconjugates incorporating the features of the double prodrugs, asdiscussed supra, and an additional moiety serving as a transportenhancer positioned between the residue of the biologically activecompound and the polymer moiety to form a 4-part structure wherein thebiologically active agent is the fourth part of the conjugate. Thisstructure provides that the residue of the biologically active compoundis optimized for transport and release substantially into a target cell.The fourth element of the “tetrapartate” is therefore the residue of thebiologically active compound, itself. Further, diagnostic tetrapartateconjugates incorporating detectable tags are also contemplated, and theuse of the terms, “tetrapartate prodrug,” or simply, “prodrug” herein,with reference to the inventive conjugates, broadly also includesconjugates and methods of making and delivering diagnostic reagents,including tagged drugs, as well, unless otherwise specified ordistinguished.

[0080] For purposes of the present invention, the terms, “biologicallyactive material,” and “biologically active compound,” and/or“biologically active agent,” etc., are used interchangeably unlessotherwise stated. These terms refer, for example, to a drug orpharmaceutical, and/or a diagnostic agent or reagent, such as adetectable label or marker. The terms “drug,” “agent,” “medicinalagent,” and “active agent” herein refer to compound(s) with usefulactivity, particularly when administered to an animal, in vivo, and/orto precursors of the same, unless otherwise stated.

[0081] As noted in the previous lines, biological activity is anyproperty of such a material or compound that is useful in an animal orperson, e.g., for medical, and/or diagnostic purposes. Preferably, thebiological activity is manifested in the intracellular space, i.e., thedrug or diagnostic agent preferably but not exclusively is useful oncedelivered/released into the cytoplasm and/or nucleus of one or moretypes of target cell of interest.

[0082] For purposes of the present invention, the use of the singular orplural is not meant to be limiting of the numerical number of thereferenced item or object. Thus, the use of the singular to refer to acell, polymer or drug does not imply that only one cell is treated, onlyone molecule is prepared or employed, and/or only one drug is employed,and the use of the plural does not exclude application to a singlereferenced item, unless expressly stated. Further to this point, forpurposes of the present invention, the terms, “cell,” “cell type,”“target cell,” and etc., are used interchangeably unless otherwisespecified and refer to both singular and plural cells, however organizedinto a tissue, tissues or other system or component, normal orpathological, of an animal or patient to be treated.

[0083] For purposes of the present invention, the term “residue” shallbe understood to mean that portion of a biologically active compoundwhich remains after it has undergone a reaction in which the prodrugcarrier portion has been attached by modification of e.g., an availablehydroxyl or amino group, to form, for example, an ester or amide group,respectively.

[0084] For purposes of the present invention, the term “alkyl” shall beunderstood to include, e.g., straight, branched, substituted C₁₋₁₂alkyls, including alkoxy, C₃₋₈ cycloalkyls or substituted cycloalkyls,etc.

[0085] When the prodrugs of the present invention include the doubleprodrugs taught by co-owned Ser. No. 09/832,557 and Ser. No. 08/992,435,it is generally preferred that the polymeric portion is first releasedby hydrolysis and then the resultant “second prodrug” moiety undergoes a1,4- or 1,6-aryl (e.g., benzyl) elimination reaction to regenerate, forexample, a moiety comprising a further prodrug, Thereafter, the releasedmoiety diffuses and/or is transported into target cells, where asubstantial proportion of the incorporated remainder of the prodrug isfurther cleaved or hydrolyzed by intracellular enzymes to release thebiologically active compound.

[0086] In addition, the terms “cancer” or “tumor” are clinicallydescriptive terms which encompass a myriad of diseases characterized bycells that exhibit unchecked and abnormal cellular proliferation. Theterm “tumor”, when applied to tissue, generally refers to any abnormaltissue growth, i.e., excessive and abnormal cellular proliferation. Theterm “cancer” is an older term which is generally used to describe amalignant tumor or the disease state arising therefrom. Alternatively,the art refers to an abnormal growth as a neoplasm, and to a malignantabnormal growth as a malignant neoplasm. These general clinical terms,when used with reference to cells, tissues, and/or one or moreconditions characterized as a disease or disorder, as used herein, areintended to be interchangeable and synonymous, unless otherwisespecified.

[0087] Broadly, the compounds according to the invention are as follows.

A. FORMULA (I)

[0088] In one aspect of the invention, there are provided compounds offormula (I):

[0089] wherein:

[0090] L₁ is a bifunctional linking moiety; for example, L₁ isindependently one of the following:

[0091] Simply by way of example, in one optional embodiment, L₁ is

[0092] In a further embodiment, Z is a transport enhancer and/orprotector that is covalently linked to D, wherein Z is selected toenhance or improve intracellular delivery of Z—[D]_(y) into a cell;relative to the intracellular delivery of D without Z.

[0093] In certain embodiments, Z optionally includes one of thefollowing: an amino acid residue, a sugar residue, a fatty acid residue,a peptide residue, a C₆₋₁₈ alkyl, a substituted aryl, a heteroaryl,—C(═O), —C(═S), and —C(═NR₁₆), and/or combinations thereof.

[0094] D is a moiety that is a leaving group, or a residue of a compoundto be delivered into a cell. Preferably but not exclusively, D is anactive biological material, or H.

[0095] The artisan will appreciate that each D can be selectedindependently, so that there can be as many as five, or more, differenttypes of moieties linked to Z for delivery into a target cell ofinterest. Preferably, D is a therapeutic agent or drug, but D is alsooptionally a diagnostic agent.

[0096] Simply by way of illustration, and in certain additional optionalembodiments, y is 2 and Z is divalent, D can be two moieties, includingboth a therapeutic agent and a diagnostic tag for delivery into the samecell type or into the cells of a tissue type of interest. Further still,such plural D moieties will comprise multiple different therapeuticagents, preferably targeted to a single type of cell, where whendelivered and released together, the different agents actsynergistically to achieve a desired therapeutic effect. In onepreferred optional embodiment, D is one or more anticancer agent(s)and/or an anticancer prodrug, or residue thereof.

[0097] In certain further embodiments, D is H, or

[0098] where

[0099] B is H, a leaving group, a residue of an amine-containing moiety,or a residue of a hydroxyl-containing moiety;

[0100] Y₁₋₆ are independently O, S or NR₁₂;

[0101] M is X or Q; where

[0102] X is an electron withdrawing group;

[0103] Q is a moiety containing a free electron pair positioned three tosix atoms from

[0104] R₁, R₄, R₇, R₈, R₉, R₁₀, R₁₂, R₁₄, R₁₅, R₁₆ and R,₁₈ areindependently selected from the group consisting of hydrogen, C₁₋₆alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, C₁₋₆ heteroalkyls and substituted C₁₋₆ heteroalkyls;

[0105] R₂, R₃, R₅ and R₆ are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₁₋₆ alkoxys, phenoxy, C₁₋₈heteroalkyls, C₁₋₈ heteroalkoxy, substituted C₁₋₆ alkyls, C₃₋₈cycloalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, halo-, nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyls andC₁₋₆ alkylcarbonyls;

[0106] Ar is a moiety which when included in Formula (I) forms amulti-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group;

[0107] (b), (m), (r), (s), (t), and (u) are independently zero or one;

[0108] (a) and (n) are independently zero or a positive integer;

[0109] (p) is zero or a positive integer;

[0110] (q) is three or four; and

[0111] R₁₁ is a polymer such as a polyalkylene oxide.

B. DESCRIPTION OF THE Ar MOIETY

[0112] Referring to Formula (I), it can be seen that the Ar is a moiety,which when included in Formula (I), forms a multi-substituted aromatichydrocarbon or a multi-substituted heterocyclic group. A key feature isthat the Ar moiety is aromatic in nature. Generally, to be aromatic, theα-electrons must be shared within a “cloud” both above and below theplane of a cyclic molecule. Furthermore, the number of α electrons mustsatisfy the Huckle rule (4n+2). Those of ordinary skill will realizethat a myriad of moieties will satisfy the aromatic requirement of themoiety and thus are suitable for use herein.

[0113] Preferred aromatic hydrocarbon moieties include, withoutlimitation:

[0114] In the above-listed aromatic moieties, J is O, S, or N—R₁₉, E andZ are independently C—R₁₉ or N—R₁₉; and R₁₉ is independently selectedfrom the same group as that which defines R₉ in Formula (I) e.g.,hydrogen, C₁₋₆ alkyls, etc. Isomers of the five and six-membered ringsare also contemplated as well as benzo- and dibenzo- systems and theirrelated congeners are also contemplated. It will also be appreciated bythe artisan of ordinary skill that the aromatic rings can optionally besubstituted with hetero-atoms such as O, S, NR₁₃, etc. so long asHuckel's rule is obeyed. Furthermore, the aromatic or heterocyclicstructures may optionally be substituted with halogen(s) and/or sidechains as those terms are commonly understood in the art. However, allstructures suitable for Ar moieties of the present invention are capableof allowing the Y₃ and C(R₁)(R₄) moieties to be in apara or an orthoarrangement with the same plane as shown in Formulas I-A and I-B, below.

[0115] wherein all variables are as defined above for Formula (I).

[0116] When the Ar moiety includes a para arrangement of the Y₃ andC(R₁)(R₄) moieties, preferred aspects of the present invention define(r), (s), (t), and (u) as one and R₂ and R₆ as being independentlyselected from the group consisting of methyl, C₁₋₆ alkyls, methyl, C₁₋₆alkoxys, and methoxy. More preferably, R₂ and R₆ are either both methylor methoxy moieties. Furthermore, R₃ and R₅ are preferably bothhydrogen, R₁ and R₄ are preferably either hydrogen, CH₃ or CH₂CH₃. Y₁through Y₄ (i.e., Y₁₋₄) are preferably O or NR₁₂ where R₁₂ is H or aC₁₋₆ alkyl or substituted alkyl. More preferably, Y₁ and Y₄ are O.

[0117] For purposes of the present invention, substituted alkyls includecarboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls andmercaptoalkyls; substituted cycloalkyls include moieties such as4-chlorocyclohexyl; aryls include moieties such as napthyl; substitutedaryls include moieties such as 3-bromophenyl; aralkyls include moietiessuch as toluyl; heteroalkyls include moieties such as ethylthiophene;substituted heteroalkyls include moieties such as 3-methoxy-thiophene;alkoxy includes moieties such as methoxy; and phenoxy includes moietiessuch as 3-nitrophenoxy. Halo- shall be understood to include fluoro,chloro, iodo and bromo.

C. LINKER MOIETY L₁

[0118] As shown above, the invention includes bifunctional linkingmoiety L₁ which when combined with

[0119] forms an amino acid residue linker, or when (p) is greater thanone, a peptide residue linker.

[0120] Suitable amino acid residues can be selected fromnaturally-occurring or synthetic, i.e. non-naturally-occurring, aminoacids including alanine, valine, leucine, isoleucine, glycine, serine,threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan,aspartic acid, glutamic acid, lysine, arginine, histidine or proline.Some preferred peptide residues include Gly-Phe-Leu-Gly and Gly-Phe-Leu.It is noted that the terminal amino group of the amino acid or peptideresidue will be proximal to R₁₁ (i.e. polymer). Peptides can be readilysynthesized or obtained from commercial sources for inclusion herein.

[0121] In alternative embodiments, L₁ includes the moiety (M) which iseither an electron withdrawing group (designated herein as X), or amoiety containing a free electron pair positioned three to six atomsfrom the

[0122] (designated herein as Q). In a particularly preferred embodiment,the tetrapartate conjugates of the invention are based upon an aromaticmoiety that is a substituted benzyl, and the first two breaks in theprodrug, in vivo, are based on a 1,4 or 1,6 benzyl eliminationmechanism. This embodiment provides a conjugate as illustrated byFIG. 1. In FIG. 1, an overview of the general reaction scheme followedin synthesis of the compounds produced in Examples 1-5, provided hereinbelow, is set forth. Precursor compound (FIG. 1, compound a) is reactedin the presence of leucine doxorubicin (“leu-dox”),dimethylaminopyridine and dimethyformamide to form (FIG. 1, compound b),wherein the variables are set forth by Table 1, below. TABLE 1 CompoundNo. Variables 2 L₁, Y₂ = 0; R₂, R₆ = H 6 L₁ = NH; Y₂ = O; R₂, R₆ = H 8L₁ = NHCOCH₂CH₂NH; Y₂ = 0; R₂, R₆ = H 10 L₁ = CH₂; Y₂ = O; R₂, R₆ = CH₃12 L₁, Y₂ = O; R₂, R₆ = CH₃

D. THE DOUBLE PRODRUG LINKAGE PORTION

[0123] The first labile bond of the tetrapartate prodrug system, whichjoins the L₁ to

[0124] is selected to hydrolyze, such as via an esterase catalyzedhydrolysis in vivo at a rate which generates sufficient amounts of the“second” prodrug compound within a suitable time after administration.The term “sufficient amounts” for purposes of the present inventionshall mean an amount which may later undergo sufficient 1,4 or 1,6-benzyl elimination in vivo to release the native compound and achieve adesired effect. Preferably, (n) is an integer from 1 to about 12. Morepreferably, (n) is 1 or 2.

1. The Electron Withdrawing Group X

[0125] In those aspects of Formula (I) where L₁ includes M, the moietymay be an electron withdrawing group, designated herein as X. Forpurposes of the present invention, “electron withdrawing groups” aregroups which tend to pull shared electrons toward themselves therebymaking carbon more electro-positive. This, in turn, destabilizes thecarbonyl moiety, causing more rapid hydrolysis. Thus, when X is in the αposition to the ester, it modulates the rates of hydrolysis andenzymatic cleavage. In particular, X can be moieties such as O, NR₂₀,

[0126] S, SO and SO₂ where Y₆ is the same as that defined by Y₁, R₁₂ andR₁₇ are the same as defined above i.e., H, C₁₋₆ alkyls, branched alkyls,aryls, etc. R₁ is the same as defined by Formula I, supra. Preferably,however, when X is NR₂₀, R₂₀ is H, a C₁₋₆ alkyl such as methyl or ethylor substituted C₁₋₆ alkyl. It is preferred that X is either O or NR₂₀.

2. Portion of L₁

[0127] Alternatively, when L₁ includes Q, which is a moiety containing afree electron pair positioned three to six atoms from the

[0128] moiety, the polymer, R₁₁, is preferably attached to Q via aheteroatom such as oxygen. In a preferred embodiment, the free electronpair is five atoms from this oxygen. Q can be selected from thenon-limiting list of C₂₋₄ alkyls or cycloalkyls, aryls or aralkyl groupssubstituted with a member of the group consisting of O, S and NR₁₂. Thefree electron pair can be anywhere along the Q moiety as long as thedefined spacing between the free electron pair and Y₄ is maintained.

[0129] In these embodiments, R₁₁ is attached to Q via NR₁₂, O, or S.Thus, Q assists hydrolysis of the prodrug linkage by anchimericassistance because the free electron pair moiety can generate a three-to six-membered, but preferably five-membered, ring by-product uponhydrolysis of the preferably ester linkage.

[0130] Q can also be selected from the group consisting of C₂₋₄ alkyls,cycloalkyls, aryls, aralkyl groups substituted with a member of thegroup consisting of NH, O, S, —CH₂—C(O)—N(H)—, and ortho-substitutedphenyls such as

[0131] wherein R₂₁ is selected from the same group as that which definesR₁₂ supra and T is any moiety linked to Q.

3. Drug Generation Via Hydrolysis of the Prodrug

[0132] The prodrug compounds of the present invention are designed sothat the t_(½) of hydrolysis is less than the t_(½) of elimination inplasma. The linkages included in the compounds have in-vivo hydrolysisrates, in plasma, that are short enough to allow sufficient amounts ofthe transport enhanced conjugate with parent compounds, i.e., the amino-or hydroxyl-containing biologically active compound, to be releasedprior to elimination. Some preferred compounds of the present invention,i.e., those in which (n) is 1, have a t_(½) for hydrolysis in plasmaranging from about 5 minutes to about 12 hours. Preferably, thecompositions have a plasma t_(½) of hydrolysis ranging from about 0.5 toabout 8 hours and most preferably from about 1 to about 6 hours.

4. 1,4 or 1,6 -Benzyl Elimination; Release of Native Drug-Linked toUptake Enhancer and Intracellular Release of Native Drug from TransportEnhancer

[0133] Once the hydrolysis of the double prodrug portion of theconjugate has taken the place in vivo, usually via esterase activity orpH moderated activity or cyclization reaction, the polymeric residue iscleaved and the resultant second prodrug moiety remains. Without meaningto be bound by any theory or hypothesis as to how the tetrapartateconjugates or prodrugs of the invention operate, it is believed thatonce the biologically effective material as linked to the uptake, entersa target cell, various intracellular peptidases and/or proteases,including e.g., cathepsins, cleave, e.g., by enzymatic hydrolysis, thetransport enhancer moiety to release the biologically effective materialwithin the target cell. The following degradation scheme is provided forillustrative purposes and is not intended to limit the scope of theinvention.

[0134] With reference to FIGS. 2A and 2B, when Ar is a benzylderivative, a 1,4 or 1,6-benzyl elimination (or an analogous reactionwith other aromatic moieties) occurs in vivo and produces the desiredtransport-enhanced native compound by electron migration, causingirreversible decomposition, which releases the transport-enhanced nativecompound.

[0135] Thus, FIG. 2A illustrates, in overview, an in vivo degradationreaction is shown wherein the variables of the starting tetrapartatecompound are defined as described supra for Formula I. With reference toFIG. 2, the illustrated tetrapartate prodrug undergoes a controllablerate cleavage, in vivo, labeled as (a) to remove R₁₁. The remainingcompound (b) immediately undergoes a fast hydrolysis (c) in the presenceof water to separate (b) and release Z—D, the enhancer-prodrug isreleased (d) mostly into the extracellular tissue space as Z—D. Z—D is,in turn, taken up by surrounding cells and hydrolyzed intracellularly(e) to release D. Any D that might be released in the intracellulartissue space is thought to be rapidly removed by blood or plasma flow,and is believed to provide little or no added benefit in terms of atherapeutic or diagnostic function.

[0136] In somewhat more detail, FIG. 2B provides schematics of in vivodegradation reactions thought to occur for compounds 14 and 17 (see,e.g., the Examples below for synthetic details). Analogously to thescheme of FIG. 2A, compound 14 is degraded by a controllable ratecleavage, in vivo, labeled as (a) to remove PEG. The remaining compound(b) immediately undergoes a fast hydrolysis (c) in the presence of waterto separate (b) and release inter alia, (d) which is analogous to Z—D ofFIG. 2A. The enhancer-doxorubicin prodrug is released mostly into theextracellular tissue space (d). The enhancer-doxorubicin prodrug is, inturn, taken up by surrounding cells and hydrolyzed intracellularly (e)to release active doxorubicin. An analogous degradation process isdescribed in FIG. 2B for compound 17, wherein the analogous processsteps are labeled as (a′), (b′), (c′), (d′) and (e′). Reaction (f) and(f′) refers to respective side reactions wherein the aromatic remainderthat is cleaved from the prodrug is converted to a water solublehydroxyl derivative.

E. SUBSTANTIALLY NON-ANTIGENIC POLYMERS

[0137] The “tetrapartate prodrug” compositions of the present inventioninclude water-soluble polymer, R₁₁. Optionally, R₁₁ includes a cappinggroup A. Capping group A includes, for example, hydrogen, C₁₋₆ alkylmoieties, carboxyalkyl, dialkyl acyl urea alkyls, and/or a compound offormula (II) shown below, which forms a bis-system:

[0138] wherein G′ is the same as D or another member of the groupdefined by D, v is 0 or 1, and the remaining variables are as set forthabove with regard to Formula (I).

[0139] Suitable examples of such polymers include polyalkylene oxidessuch as polyethylene glycols which are also preferably substantiallynon-antigenic. The general formula for PEG and its derivatives, i.e.A′—O—(CH₂CH₂O)_(x)—(CH₂)_(n)—A, where (x) represents the degree ofpolymerization (i.e. 10-2,300) or number of repeating units in thepolymer chain and is dependent on the molecular weight of the polymer;(n) is zero or a positive integer; A is a capping group as definedherein, i.e. an —H, amino, carboxy, halo, C₁₋₆ alkyl or other activatinggroup and A′ is the same as A or another A moiety. Also useful arepolypropylene glycols, branched PEG derivatives such as those describedin commonly-assigned U.S. Pat. No. 5,643,575, “star-PEG's” andmulti-armed PEG's such as those described in Shearwater Polymers, Inc.catalog “Polyethylene Glycol Derivatives 1997-1998”; the disclosure ofeach is incorporated herein by reference. It will be understood that thewater-soluble polymer will be functionalized for attachment to thelinkage via M, X or Q herein. As an example, the PEG portion of theprodrugs can be the following non-limiting compounds:

[0140] —C(═Y₇)—(CH₂)_(n)—O—(CH₂CH₂O)_(x)—A,

[0141] —C(═Y₇)—Y₇—(CH₂)_(n)—O—(CH₂CH₂O)_(x)—A and

[0142] —C(═Y₇)—NR₁₂—(CH₂)_(n)—O—(CH₂CH₂O)_(x)—A,

[0143] where Y₇ is O or S; and A, R₁₂, (n) and (x) are as defined above.

[0144] In many aspects of the present invention, polyethylene glycols(PEG's), mono-activated, C₁₋₄ alkyl-terminated PAO's such asmono-methyl-terminated polyethylene glycols (mPEG's) are preferred whenmono-substituted polymers are desired; bis-activated polyethylene oxidesare preferred when di-substituted prodrugs are desired.

[0145] In order to provide the desired hydrolyzable linkage, mono- ordi-acid activated polymers such as PEG acids or PEG diacids can be usedas well as mono-or di-PEG amines and mono- or di-PEG diols. Suitable PAOacids can be synthesized by first converting mPEG—OH to an ethyl esterfollowed by saponification. See also Gehrhardt, H., et al. PolymerBulletin 18: 487 (1987) and Veronese, F. M., et al., J. ControlledRelease 10; 145 (1989). Alternatively, the PAO-acid can be synthesizedby converting mPEG—OH into a t-butyl ester followed by acid cleavage.See, for example, commonly assigned U.S. Pat. No. 5,605,976. Thedisclosures of each of the foregoing are incorporated by referenceherein.

[0146] Although PAO's and PEG's can vary substantially in number averagemolecular weight, polymers ranging from about 2,000 to about 100,000daltons are usually selected for the purposes of the present invention.Molecular weights of from about 20,000 to about 50,000 are preferred,and 20,000 to about 40,000 are particularly preferred. The numberaverage molecular weight of the polymer selected for inclusion in the“tetrapartate prodrug” must be sufficient so as to provide sufficientcirculation of the “tetrapartate prodrug” before removal of thetransport enhancer. Within the ranges provided above, polymers havingmolecular weight ranges of at least 20,000 are preferred in some aspectsfor chemotherapeutic and organic moieties. In the case of somenucleophiles such as certain proteins, enzymes and the like, polymershaving a molecular weight range of from about 12,000 to about 20,000 arepreferred.

[0147] The polymeric substances included herein are preferablywater-soluble at room temperature. A non-limiting list of such polymersinclude polyalkylene oxide homopolymers such as polyethylene glycol(PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymersthereof and block copolymers thereof, provided that the water solubilityof the block copolymers is maintained.

[0148] As an alternative to PAO-based polymers, effectivelynon-antigenic materials such as dextran, polyvinyl alcohols,carbohydrate-based polymers, hydroxypropylmethacrylamide (HPMA), andcopolymers thereof, etc. and the like can be used if the same type ofactivation is employed as described herein for PAO's such as PEG. Thoseof ordinary skill in the art will realize that the foregoing list ismerely illustrative and that all polymeric materials having thequalities described herein are contemplated. For purposes of the presentinvention, “effectively non-antigenic” means all polymeric materialsunderstood in the art as being nontoxic and not eliciting an appreciableimmune response in mammals.

F. POLYMERIC TETRAPARTATE TRANSPORT SYSTEM SYNTHESIS

[0149] Synthesis of representative, specific prodrugs is set forth inthe Examples. Generally, however, the transport enhanced prodrugs of thepresent invention can be prepared in several fashions. Thus, one methodincludes reacting a compound of formula

[0150] with a compound of the formula (IV): Lx—Z—[D]_(y)

[0151] In this embodiment, L₁ is a bifunctional linking moiety:

[0152] B and Lx are independently selected leaving groups and aredefined as above for when D is a leaving group;

[0153] Z is a moiety that is actively transported into a target cell, ahydrophobic moiety and combinations thereof. Preferably, Z is selectedfrom the group consisting of an amino acid residue, a C₆₋₁₈ alkyl, asubstituted aryl, a hetero aryl, —C(═Y₄), —C(═S), —C(═NR₁₆) andcombinations thereof, wherein R₁₆ is selected from the same group asR₁₂;

[0154] R₁, R₄, R₉, R₁₀ and R₁₂ are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls and substitutedC₁₋₆ heteroalkyls;

[0155] R₂, R₃, R₅ and R₆ are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₁₋₆ alkoxys, phenoxy, C₁₋₈heteroalkyls, C₁₋₈ heteroalkoxys, substituted C₁₋₆ alkyls, C₃₋₈cycloalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, halo-, nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyls andC₁₋₆ alkylcarbonyls;

[0156] Ar is a moiety which when included in Formula (III), andsubsequently in Formula (I), forms a multi-substituted aromatichydrocarbon or a multi-substituted heterocyclic group;

[0157] (m), (r), (s), (t), and (u) are independently zero or one;

[0158] (p) is zero or a positive integer;

[0159] Y₁₋₄ are independently O, S, or NR₁₂, wherein the definition ofR₁₂ is defined as per formula I, supra; (y) is one or two; and R₁₁ is amonovalent or divalent polymer residue.

[0160] Typically, the reaction between (III) and (IV) is conducted inthe presence of a solvent and a base. The solvent is preferably an inertsolvent, i.e., inert with respect to the reactants and products.Exemplary solvents include, simply by way of example, chloroform,toluene, methylene chloride, dimethylformamide and combinations thereof.Generally, dimethylformamide is preferred. Exemplary bases solventsinclude, simply by way of example, dimethylaminopyridine,diisopropylethylamine, pyridine, triethylamine and combinations thereofGenerally, dimethylaminopyridine is preferred.

[0161] Optionally, the tetrapartate prodrugs of the invention can alsobe prepared by reacting a compound of formula (V)

[0162] with a biologically effective material, e.g., a biologicallyactive compound such as a drug or diagnostic tag. In this embodiment,

[0163] La is a leaving group for Formula V as defined for D when D is aleaving group.

[0164] L₁ is a bifunctional linking moiety;

[0165] Z is a moiety that is actively transported into a target cell, ahydrophobic moiety and combinations thereof Preferably, Z is selectedfrom the group consisting of an amino acid residue, a C₆₋₁₈ alkyl, asubstituted aryl, a hetero aryl, —C(═Y₄), —C(═S), —C(═NR₁₆) andcombinations thereof;

[0166] R₁, R₄, R₉, R₁₀, R₁₂ and R₁₆ are independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls and substitutedC₁₋₆ heteroalkyls;

[0167] R₂, R₃, R₅ and R₆ are independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyls, C₁₋₆ alkoxys, phenoxy, C₁₋₈heteroalkyls, C₁₋₈ heteroalkoxys, substituted C₁₋₆ alkyls, C₃₋₈cycloalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, halo-, nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyls andC₁₋₆ alkylcarbonyls.

[0168] Ar is a moiety which when included in Formula (V) forms amulti-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group.

[0169] (m), (r), (s), (t), and (u) are independently zero or one.

[0170] (p) is zero or a positive integer.

[0171] Y₁₋₄ are independently O, S, or NR₁₂, wherein the definition ofR₁₂ is defined as per formula I, supra; and R₁₁ is a monovalent ordivalent polymer residue.

[0172] Optionally, the reaction between (V) and the biologicallyeffective material is conducted in the presence of a coupling agent,including, for example, 1,3-diiso-propylcarbodiimide, a dialkylcarbodiimides, 2-halo-1-alkyl-pyridinium halide,1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, 1-propanephosphonicacid cyclic anhydride, phenyl dichlorophosphates, and combinationsthereof. In addition, the reaction between (V) and the biologicallyactive material is typically conducted in the presence of a solvent anda base, each of which are defined as described supra, for the reactionbetween formulas (III) and (IV). In addition, the base is preferablydimethylamino pyridine.

[0173] Biologically effective materials for the tetrapartate prodrug arediscussed below.

G. LEAVING GROUPS OR RESIDUE PORTION “D”

[0174] 1. Leaving Groups

[0175] In those aspects where B is a leaving group and further withreference to the Lz and/or La leaving groups, as described above,suitable leaving groups include, without limitations, moieties such asN-hydroxybenzotriazolyl, halogen, N-hydroxyphthalimidyl, p-nitrophenoxy,imidazolyl, N-hydroxysuccinimidyl; thiazolidinyl thione, or other goodleaving groups as will be apparent to those of ordinary skill. Thesynthesis reactions used and described herein will be understood bythose of ordinary skill without undue experimentation.

[0176] For example, the acylated intermediate compound (III) can bereacted with such as 4-nitrophenyl chloroformate, disuccinimidylcarbonate (DSC), carbonyldiimidazole, thiazolidine thione, etc. toprovide the desired activated derivative.

[0177] The acylation of the p-hydroxybenzyl alcohol or the p-aminobenzylalcohol and the o-hydroxbenzyl alcohol or the o-aminobenzyl alcohol canbe carried out with, for example, thiazolidine thione activatedpolymers, succinimidyl carbonate activated polymers, carboxylic acidactivated polymers, blocked amino acid derivatives.

[0178] Once in place, the “activated” form of the PEG prodrug (orblocked prodrug) is ready for conjugation with an amine- orhydroxyl-containing compound. Some preferred activated transport formsare shown below.

[0179] 2. Residues of Biologically Active Materials

[0180] Broadly, the only limitations on the types of biologicallyeffective materials suitable for inclusion herein is that there isavailable at least one site for covalent attachment to the uptakeenhancer moiety. Simply by way of example, this can be a (primary orsecondary) amine-containing position or functional group which can reactand link with a carrier portion, e.g., by forming an amide bond. Othersites for covalent attachment to the uptake enhancer moiety include,e.g., a hydroxyl functional group, to form, e.g., an ester linkage. Ofcourse, the artisan will appreciate that the selected linkage betweenthe biologically effective material of interest, and the uptake enhanceris such that there is no substantial loss of bioactivity after thedouble prodrug portion of the conjugate releases the parent compound inlinkage with a transport enhancer.

[0181] After conjugation, the remaining amine-containing compound isreferred to as the residue of the unconjugated compound.

I. Residues of Amine-containing Compounds

[0182] In some aspects of the invention, e.g., after the prodrugtransport has been formed, D is a residue of an amine-containing organiccompound. Organic compounds include, without limitation, moieties suchas anthracycline compounds including daunorubicin, doxorubicin;p-aminoaniline mustard, melphalan, Ara-C (cytosine arabinoside) andrelated anti-metabolite compounds, e.g., gemcitabine, etc.

[0183] Also included herein is any portion of a polypeptide, nucleicacid, peptide nucleic acids and any combinations thereof, ranging insize from about 50 daltons through about 2,500 daltons, or greater,demonstrating in vivo bioactivity. This includes, e.g., peptides,nucleic acids (DNA, RNA) with at least one amine functional group, e.g.,peptide nucleic acids and the like.

[0184] Thus, in a preferred aspect of the invention, biologicallyeffective material is a biologically active compound that is suitablefor medicinal or diagnostic use in the treatment of animals, e.g.,avians and/or mammals, including humans, for conditions for which suchtreatment is desired. The foregoing list of biological materials ismeant to be illustrative and not limiting for the compounds which can bemodified. Those of ordinary skill will realize that other such compoundscan be similarly modified without undue experimentation. It is to beunderstood that those biologically active materials not specificallymentioned but having suitable amino-groups are also intended and arewithin the scope of the present invention.

II. Residues of Hydroxyl-Containing Compounds

[0185] a. Camptothecin and Related Topoisomerase I Inhibitors

[0186] Camptothecin is a water-insoluble cytotoxic alkaloid produced byCamptotheca accuminata trees indigenous to China and nothapodytesfoetida trees indigenous to India. Camptothecin and related compoundsand analogs are also known to be potential anticancer or antitumoragents and have been shown to exhibit these activities in vitro and invivo. Camptothecin and related compounds are also candidates forconversion to the tetrapartate prodrugs of the present invention.

[0187] Camptothecin and certain related analogues share the structure:

[0188] From this core structure, several known analogs have beenprepared. For example, the A ring in either or both of the 10- and11-positions can be substituted with an OH. The A ring can also besubstituted in the 9-position with a straight or branched C₁₋₃₀ alkyl orC₁₋₁₇ alkoxy, optionally linked to the ring by a heteroatom i.e. —O orS. The B ring can be substituted in the 7-position with a straight orbranched C₁₋₃₀ alkyl or substituted alkyl-, C₅₋₈ cycloalkyl, C₁₋₃₀alkoxy, phenyl alkyl, etc., alkyl carbamate, alkyl carbazides, phenylhydrazine derivatives, amino-, aminoalkyl-, aralkyl, etc. Othersubstitutions are possible in the C, D and E rings.

[0189] See, for example, U.S. Pat. Nos. 6,111,107; 5,004,758; 4,943,579;Re 32,518, the contents of which are incorporated herein by reference.Such derivatives can be made using known synthetic techniques withoutundue experimentation. Preferred camptothecin derivatives for use hereininclude those which include a 20—OH or another OH moiety which iscapable of reacting directly with activated forms of the polymertransport systems described herein or to the linking moietyintermediates, e.g. iminodiacetic acid, etc., which are then attached toa polymer such as PEG.

[0190] Reference to camptothecin analogs herein has been made forpurposes of illustration and not limitation.

[0191] b. Taxanes and Paclitaxel Derivatives

[0192] One class of compounds included in the tetrapartate prodrugcompositions of the present invention is taxanes. For purposes of thepresent invention, the term “taxane” includes all compounds within thetaxane family of terpenes. Thus, taxol (paclitaxel), 3′-substitutedtert-butoxy-carbonyl-amine derivatives (taxoteres) and the like as wellas other analogs which are readily synthesized using standard organictechniques or are available

[0193] from commercial sources such as Sigma Chemical of St. Louis,Miss. are within the scope of the present invention. Representativetaxanes are shown below.

Paclitaxel: R′₁═C₆H₅; R′₂═CH₃CO; Taxotere: R′₁═(CH₃)₃CO; R′₂═H

[0194] These derivatives have been found to be effective anti-canceragents. Numerous studies indicate that the agents have activity againstseveral malignancies. To date, their use has been severely limited by,among other things, their short supply, poor water solubility andhypersensitivity. It is to be understood that other taxanes includingthe 7-aryl-carbamates and 7-carbazates disclosed in commonly assignedU.S. Pat. Nos. 5,622,986 and 5,547,981 can also be included in thetetrapartate prodrugs of the present invention. The contents of theforegoing U.S. patents are incorporated herein by reference. The onlylimitation on the taxane is that it must be capable of undergoing ahydroxyl based substitution reaction such as at the 2′ position.Paclitaxel, however, is a preferred taxane.

[0195] c. Additional Biologically-Active Moieties

[0196] In addition to the foregoing molecules, the tetrapartate prodrugformulations of the present invention can be prepared using many othercompounds. For example, drugs such as gemcitabine, etoposide,triazole-based antifungal agents such as fluconazole and/or ciclopiroxcan be used.

[0197] The parent compounds selected for tetrapartate prodrug forms neednot be substantially water-insoluble, although the polymer-basedtetrapartate prodrugs of the present invention are especially wellsuited for delivering such water-insoluble compounds. Other usefulparent compounds include, for example, certain low molecular weightbiologically active proteins, enzymes and peptides, including peptidoglycans, as well as other anti-tumor agents; cardiovascular agents suchas forskolin; anti-neoplastics such as combretastatin, vinblastine,doxorubicin, ara-C, maytansine, etc.; anti-infectives such asvancomycin, erythromycin, etc.; anti-fungals such as nystatin,amphoteracin B, triazoles, papulocandins, pneumocandins, echinocandins,polyoxins, nikkomycins, pradimicins, benanomicins, etc. see,“Antibiotics That Inhibit Fungal Cell Wall Development” Annu. Rev.Microbiology, 1994, 48:471-97, the contents of which are incorporatedherein by reference; anti-anxiety agents, gastrointestinal agents,central nervous system-activating agents, analgesics, fertility orcontraceptive agents, anti-inflammatory agents, steroidal agents,anti-urecemic agents, cardiovascular agents, vasodilating agents,vasoconstricting agents and the like.

[0198] After conjugation, the remaining amine-or hydroxyl-containingcompound is referred to as the residue of the unconjugated compound.

3. Polymeric Hybrids

[0199] In another aspect of the invention there are provided hybridtypes of the polymeric tetrapartate prodrug transport system describedherein. In particular, the hybrid system includes not only thereversible double prodrug system described above but also a secondpolymeric transport system based on more permanent types of linkages.The hybrids can be prepared by at least two methods. For example, thebenzyl-elimination-based prodrug can be synthesized first and thenPEGylated using any art-recognized activated polymer such asthiazolidinyl thione-or succinimidyl carbonate-activated PEG.Alternatively, the more permanent conjugation reaction can be performedfirst and the resultant conjugates can be used to form the doubleprodrug portion of the tetrapartate conjugates described herein. It willbe understood that the hybrid systems will be better suited forproteins, enzymes and the like where multiple amino groups are availablefor attachment of the polymeric transport forms. For purposes of thepresent invention, “activated polymers” will be understood to includepolymers containing one or more terminal groups which are capable ofreacting with one or more of α-amino groups, ε-amino groups, histidinenitrogens, carboxyl groups, sulfhydryl groups, etc. found on enzymes,proteins, etc., as well as such groups found on synthetically preparedorganic compounds. It will further be appreciated that the activatinggroups described below can also be used to form the activated transportforms described above.

[0200] The activating terminal moiety can be any group which facilitatesconjugation of the polymers with the biologically active material, i.e.,protein, enzyme, etc. either before of after the double prodrugtransport system of the present invention has been synthesized. See, forexample, U.S. Pat. No. 4,179,337, the disclosure of which is herebyincorporated by reference. Such activating groups can be a moietyselected from:

[0201] I. Functional groups capable of reacting with an amino group suchas:

[0202] a) carbonates such as the p-nitrophenyl, or succinimidyl; see,for example, U.S. Pat. No. 5,122,614, the disclosure of which is herebyincorporated by reference;

[0203] b) carbonyl imidazole;

[0204] c) azlactones; see, for example, U.S. Pat. No. 5,321,095, thedisclosure of which is hereby incorporated by reference;

[0205] d) cyclic imide thiones see, for example, U.S. Pat. No.5,349,001, the disclosure of which is hereby incorporated by reference;

[0206] e) isocyanates or isothiocyanates; or

[0207] f) active esters such as N-hydroxy-succinimidyl orN-hydroxybenzotriazolyl.

[0208] II. Functional groups capable of reacting with carboxylic acidgroups and reactive carbonyl groups such as:

[0209] a) primary amines; or

[0210] b) hydrazine and hydrazide functional groups such as the acylhydrazides, carbazates, semicarbamates, thiocarbazates, etc.

[0211] III. Functional groups capable of reacting with mercapto orsulfhydryl groups such as phenyl glyoxals; see, for example, U.S. Pat.No. 5,093,531, the disclosure of which is hereby incorporated byreference;

[0212] IV. Functional groups capable of reacting with hydroxyl groupssuch as (carboxylic) acids or other nucleophiles capable of reactingwith an electrophilic center. A non-limiting list includes, for example,hydroxyl, amino, carboxyl, thiol groups, active methylene and the like.

[0213] The activating moiety can also include a spacer moiety locatedproximal to the polymer. The spacer moiety may be a heteroalkyl, alkoxy,alkyl containing up to 18 carbon atoms or even an additional polymerchain. The spacer moieties can added using standard synthesistechniques.

H. METHODS OF TREATMENT

[0214] Broadly, another aspect of the present invention provides methodsfor delivering biologically active materials, such as therapeutic ordiagnostic agents into cells where such biological activity is desired.While the tetrapartate prodrugs of the invention are readily employed todeliver biologically active materials into a wide variety of cells,found throughout the animal body, certain applications are preferred.For example, the tetrapartate prodrugs of the invention are particularlyuseful in delivering biologically active materials, such as drugs and/ordiagnostics, into cells present in tissues exhibiting theabove-discussed EPR effect. A number of tissue types exhibiting EPRoccur in different diseases and disorders, including tissues undergoinginflammation, toxic reactions of various kinds, as well as solid tumors.

[0215] Thus, the broad method includes contacting living tissue with theinventive tetrapartate prodrugs. Preferably the tissue exhibits the EPReffect, so that polymer linked conjugates preferably enter such tissues.Of course, the artisan will appreciate that an agent, once deliveredinto a target cell and activated, can then be released by that cell andprovide biological activity in other tissue spaces.

[0216] Simply by way of example, a non-active prodrug of the inventionthat is delivered into an exocrine cell of the liver or pancreas undersuitable conditions, e.g., during a disease process that causesinflammation, and results in an EPR effect, can be activated within thecytoplasm of the target cell, and then the activated drug or diagnosticagent can then be secreted into the gastrointestinal (“G.I”) tract fluidspace for therapeutic and/or diagnostic purposes. In this instance,treatment and/or diagnosis of certain diseases or disorders of the G.I.tract by means of the targeted delivery of appropriate agents, includinganti-cancer or antiviral agents, is therefore facilitated. Analogousmethods of treatment and delivery of biologically active materials isreadily contemplated for other organ and/or tissue systems.

[0217] In one preferred embodiment, the tissues are tumor or cancertissues, and the tetrapartate prodrugs of in the invention compriseagents suitable for treatment and/or diagnosis of such conditions. Thus,the tetrapartate prodrug compositions are useful for, among otherthings, treating diseases which are similar to those which are treatedwith the parent compound(s), e.g., including compounds suitable fortreating neoplastic disease, reducing tumor burden, inhibitingmetastasis of tumors or neoplasms and preventing recurrences oftumor/neoplastic growths in mammals. The treated animals are preferablymammals, and more preferably human patients. While veterinary use of theprodrugs of the invention will typically be employed in mammalianspecies, it is further contemplated that the prodrugs can also bereadily employed in other species generally within the veterinarypractice and animal husbandry arts, e.g., including highly valuednon-mammalian exotic animals.

[0218] The amount of the prodrug and/or diagnostic tetrapartate tag thatis administered will depend upon the amount of the parent moleculeincluded therein. Generally, the amount of tetrapartate prodrug used inthe treatment methods is that amount which effectively achieves thedesired therapeutic or diagnostic result in mammals. Naturally, thedosages of the various prodrug compounds will vary somewhat dependingupon the parent compound, rate of in vivo hydrolysis, molecular weightof the polymer, etc. In general, tetrapartate prodrug polymericderivatives are administered in amounts ranging from about 5 to about500 mg/m₂ per day, based on the native drug. The range set forth aboveis illustrative and those skilled in the art will determine the optimaldosing of the prodrug selected based on clinical experience and thetreatment indication. Actual dosages will be apparent to the artisanwithout undue experimentation.

[0219] The compositions, including prodrugs, of the present inventioncan be included in one or more suitable pharmaceutical compositions foradministration to an animal in need thereof. The pharmaceuticalcompositions may be in the form of a solution, suspension, tablet,capsule or the like, prepared according to methods well known in theart. It is also contemplated that administration of such compositionsmay be by the oral and/or parenteral routes depending upon the needs ofthe artisan. A solution and/or suspension of the composition may beutilized, for example, as a carrier vehicle for injection orinfiltration of the composition by any art known methods, e.g., byintravenous, intramuscular, subdermal injection and the like.

[0220] Such administration may also be by infusion into a body space orcavity, as well as by inhalation and/or intranasal routes. In preferredaspects of the invention, however, the prodrugs are parenterallyadministered to animals in need thereof.

[0221] The novel methods of treatment or administration according to theinvention further includes the multi-step cleavage of the prodrug,resulting in release of the biologically active material, such as a drugor tag, within a target cell.

I. IN VIVO DIAGNOSTICS

[0222] A further aspect of the invention provides the tetrapartateconjugates of the invention optionally prepared with a diagnostic taglinked to the transport enhancer described above, wherein the tag isselected for diagnostic or imaging purposes. Thus, a suitable tag isprepared by linking any suitable moiety, e.g., an amino acid residue, toany art-standard emitting isotope, radio-opaque label, magneticresonance label, or other non-radioactive isotopic labels suitable formagnetic resonance imaging, fluorescence-type labels, labels exhibitingvisible colors and/or capable of fluorescing under ultraviolet, infraredor electrochemical stimulation, to allow for imaging tumor tissue duringsurgical procedures, and so forth. Optionally, the diagnostic tag isincorporated into and/or linked to a conjugated therapeutic moiety,allowing for monitoring of the distribution of a therapeuticbiologically active material within an animal or human patient.

[0223] In a still further aspect of the invention, the inventive taggedtetrapartate conjugates are readily prepared, by art-known methods, withany suitable label, including, e.g., radioisotope labels. Simply by wayof example, these include ¹³¹Iodine, ¹²⁵Iodine, ^(99m)Technetium and/or¹¹¹Indium to produce radioimmunoscintigraphic agents for selectiveuptake into tumor cells, in vivo. For instance, there are a number ofart-known methods of linking peptide to Tc-99m, including, simply by wayof example, those shown by U.S. Pat. Nos. 5,328,679; 5,888,474;5,997,844; and 5,997,845, incorporated by reference herein.

[0224] Broadly, for anatomical localization of tumor tissue in apatient, the tetrapartate conjugate tag is administered to a patient oranimal suspected of having a tumor. After sufficient time to allow thelabeled immunoglobulin to localize at the tumor site(s), the signalgenerated by the label is detected, for instance, visually, by X-rayradiography, computerized transaxial tomography, MRI, by instrumentaldetection of a luminescent tag, by a photo scanning device such as agamma camera, or any other method or instrument appropriate for thenature of the selected tag.

[0225] The detected signal is then converted to an image or anatomicaland/or physiological determination of the tumor site. The image makes itpossible to locate the tumor in vivo and to devise an appropriatetherapeutic strategy. In those embodiments where the tagged moiety isitself a therapeutic agents, the detected signal provides evidence ofanatomical localization during treatment, providing a baseline forfollow-up diagnostic and therapeutic interventions.

J. EXAMPLES

[0226] It should be noted for all of the compounds that were produced bythe following examples, and as illustrated by FIGS. 1-9, that “PEG” is:

[0227] although other art-known variations are readily employed, asmentioned supra. In addition, the PEG employed in the following exampleshad a molecular weight of about 40 kDa.

[0228] The following examples serve to provide further appreciation ofthe invention but are not meant in any way to restrict the effectivescope of the invention.

Example 1 Synthesis of Compound 2

[0229] Compound 2 (FIG. 3) was prepared by one of the following methods:

Method 1

[0230] Leucine-doxorubicin (130 mg, 0.198 mmol) anddimethylaminopyridine (“DMAP,” 121 mg, 0.99 mmol) were added to asolution of compound 1, having a PEG chain of about 40 kDa (2.4 g, 0.060mmol, FIG. 1) in 40 mL of anhydrous dimethyformamide (“DMF”). Themixture was stirred at room temperature overnight. Ethyl ether (˜200 mL)was added to the reaction mixture to precipitate PEG derivatives, andthe solid filtered and recrystallized twice from from 80 mL 2-propanol(“IPA”) to give pure product of compound 2 (1.85 g, 75%, FIG. 3).

Method 2

[0231] (Step 1) To a solution of compound 1 (4.5 g, 0.111 mmol, FIG. 4)in 80 mL of anhydrous methylene chloride, was added leucine t-butylester (248 mg, 1.11 mmol) and DMAP (136 mg, 1.11 mmol). The reactionmixture was stirred for 18 hours at room temperature. The mixture wasevaporated under reduced pressure, and the residue was re-crystallizedfrom IPA to give compound 3 (4.5 g, 99%, FIG. 4). The structure wasconfirmed by ¹³C NMR (67.8 MHz, CDCl₃) with peaks at δ 171.55, 155.24,152.88, 150.24, 133.79, 128.68, 120.48, 81.05, 69.0-72.5 (PEG), 52.51,41.26, 27.40, 24.19, 22.27, 21.44.

[0232] (Step 2) compound 3 (4.70 g, 0.114 mmol) was dissolved in 22.5 mLtrifluoroacetic acid and 45 mL of methylene chloride and stirred at roomtemperature for 2 hours. Ethyl ether was added to precipitate the PEGderivative. The crude product was filtered and washed with ethyl etherto yield compound 4 (4.4 g, 94%, FIG. 4). The structure was confirmed by¹³C NMR (67.8 MHz, CDCl₃) with peaks at δ 173.30, 155.36, 152.94,150.24, 133.81, 128.67, 120.52, 69.0-72.5 (PEG), 52.65, 41.20, 24.18,22.44, 21.31.

[0233] (Step 3) Doxorubicin (57 mg, 0.0984 mmol) and DMAP (42 mg, 0.344mmol) were added to a solution of compound 4 (1.0 g, 0.0246 mmol, FIG.4) in anhydrous methylene chloride (20 mL) at 0° C. for 20 minutes.1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (“EDC,”28 mg, 0.148 mmol) was added, and the reaction mixture gradually warmedto room temperature and stirred overnight. The solution was filtered bygravity, and the solvent evaporated. The residual solid wasrecrystallized from IPA (50 mL) to give pure compound 2 (0.656 g, 67%).

[0234] The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃) withpeaks at δ 213.56, 186.82, 186.50, 171.27, 160.76, 155.93, 155.50,153.21, 150.51, 135.55, 135.22, 133.84, 133.47, 129.05, 120.81, 119.53,118.24, 111.26, 111.00, 100.40, 69.0-72.5 (PEG), 68.43, 67.46, 67.16,65.86, 56.41, 53.39, 45.15, 41.40, 35.23, 29.21, 24.35, 22.72, 21.57,16.61.

Example 2 Synthesis of Compound 6

[0235] Compound 6 (FIG. 5), was prepared by adding leucine-doxorubicin(130 mg, 0.198 mmol, FIG. 3) and DMAP (121 mg, 0.99 mmol) to a solutionof compound 5 (2.2 g, 0.054 mmol, FIG. 5) in anhydrous DMF (30 mL).After stirring at room temperature overnight, ethyl ether (˜100 mL) wasadded to the reaction mixture and the precipitated solid filtered. Thesolid was recrystallized twice from IPA (70 mL) to give pure product(2.09 g, 93%).

[0236] The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃) withpeaks at: δ 213.40, 186.56, 186.23, 171.23, 160.61, 155.83, 155.65,155.23, 154.25, 150.51, 135.41, 134.99, 133.43, 133.25, 132.79, 128.85,121.26, 120.40, 119.36, 118.17, 111.06, 110.88, 100.34, 69.0-72.5 (PEG),68.33, 67.10, 65.89, 65.08, 56.28, 53.27, 45.03, 41.20, 40.61, 40.41,35.25, 33.50, 29.07, 24.23, 22.65, 21.40, 16.52.

Example 3 Synthesis of Compound 8

[0237] Compound 8 (FIG. 6), was prepared by the methods described inExample 2, above: by adding leucine-doxorubicin (80 mg, 0.122 mmol ) toa solution of compound 7 (1.6 g, 0.41 mmol, FIG. 4) in 30 mL ofanhydrous DMF, and reacting the mixture to give the product 8, (1.44 g,85%) which was recrystallized from IPA.

[0238] The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃) withpeaks at: δ 213.64, 186.80, 186.42, 171.32, 171.21, 160.76, 155.97,155.41, 154.30, 150.68, 135.54, 135.19, 133.50, 133.38, 129.06, 127.52,121.41, 119.54, 118.25, 111.26, 111.06, 100.47, 69.0-72.5 (PEG), 68.54,67.20, 66.13, 65.25, 56.43, 53.48, 45.11, 41.24, 38.97, 37.10, 35.40,35.06, 33.64, 29.25, 24.39, 22.75, 21.55, 16.64.

Example 4 Synthesis of Compound 10

[0239] Compound 10 (FIG. 7A) was prepared by the methods described inExample 2, above: by adding leucine-doxorubicin (97 mg, 0.147 mmol, FIG.7A) to a solution of compound 9 (2.0 g, 0.049 mmol, FIG. 7A) in 30 mL ofanhydrous DMF. Pure compound 10 was obtained by recrystallization fromIPA (1.70 g, 83%).

[0240] The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃) withpeaks at: δ 213.71, 186.93, 186.55, 171.35, 168.14, 160.88, 156.03,155.51, 135.61, 135.31, 133.67, 133.50, 130.08, 128.38, 120.69, 119.64,118.31, 111.38, 111.19, 100.50, 69.0-72.5 (PEG), 68.64, 67.13, 66.28,65.33, 56.51, 53.60, 45.23, 41.43, 35.50, 33.80, 29.47, 24.49, 22.79,21.68, 16.69.

Example 5 Synthesis of Compound 12

[0241] Compound 12 (FIG. 7B), was prepared by the methods described inExample 2, above: by adding leucine-doxorubicin (134 mg, 0.204 mmol,FIG. 7B) to a solution of compound 11 (2.0 g, 0.049 mmol) ) in 30 mL ofanhydrous DMF. Compound 12 was purified by recrystallization from IPA(1.69 g, 82%).

Example 6 Synthesis of Compound 14

[0242] Compound 14 (FIG. 8), was prepared as follows.

[0243] (Step 1) To a solution of compound 1 (6.88 g, 0.170 mmol, FIG.8), in 90 mL anhydrous DMF, was added 12-aminododecanoic acid (0.15 g,0.680 mmol) and DMAP (0.114 g, 0.934 mmol). The resulting reactionmixture was stirred for 18 hours at a temperature between 50 to 60° C.The mixture was filtered, the filtrate evaporated under reducedpressure, and the residue recrystallized from IPA to give compound 13(6.2 g, 90). The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃)with peaks at δ 174.58, 155.79, 152.97, 150.20, 134.19, 128.77, 127.31,120.52, 69.0-72.5 (PEG), 40.54, 33.32, 29.42, 28.90, 28.70, 28.59,26.17, 24.87, 24.36.

[0244] (Step 2) To a solution of compound 13 (3.0 g, 0.074 mmol) asobtained in Step 1, above, doxorubicin hydrochloride (256 mg, 0.441mmol), 4-methylmorpholine (“NMM”, 130 uL, 1.18 mmol), and1-hydroxybenzotriazole hydrate (“HOBT”, 60 mg, 0.441 mmol) in 80 mLanhydrous DMF/methylene chloride (1:1) was EDC (113 mg, 0.589 mmol), andthe mixture stirred at room temperature overnight. The solvent wasevaporated under reduced pressure and the residue was recrystallizedfrom IPA (100 mL) to give pure compound 14 (2.80 g, 91%)).

[0245] The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃), withpeaks at δ 213.40, 186.56, 186.18, 172.11, 160.56, 155.97, 155.83,155.22, 153.04, 150.26, 135.41, 134.96, 134.23, 133.45, 133.26, 128.83,127.38, 120.60, 120.38, 118.14, 111.00, 110.81, 100.50, 69.0-72.5 (PEG),68.28, 67.28, 67.04, 65.89, 65.02, 56.26, 44.61, 40.60, 40.41, 35.88,35.26, 33.43, 29.46, 29.30, 28.93, 28.86, 26.20, 25.12, 16.55.

Example 7 Synthesis of Compound 17

[0246] Compound 17 was prepared as illustrated by FIG. 9.

[0247] (Step 1) To a solution of compound 13 (5.20 g, 0.128 mmol) in 50mL anhydrous methylene chloride was added leucine t-butyl ester (0.287g, 1.28 mmol), DMAP (0.281 g, 2.30 mmol), and EDC (0.197 g, 1.02 mmol).The reaction mixture was stirred at room temperature for 18 hours.Solvent was removed under reduced pressure, and the residuerecrystallized from IPA to yield 15 (4.8 g, 92%, FIG. 9). The structurewas confirmed by ¹³C NMR (67.8 MHz, CDCl₃) with peaks at δ 172.22,171.87, 155.79, 152. 98, 150.22, 134.20, 128.78, 120.54, 69.0-72.5(PEG), 50.62, 41.37, 40.55, 35.96, 29.43, 28.88, 28.73, 27.49, 26.19,25.05, 24.89, 24.39, 22.32, 21.65.

[0248] (Step 2) compound 15 (4.70 g, 0.114 mmol) was dissolved in 25 mLtrifluoroacetic acid and 50 mL methylene chloride and stirred at roomtemperature for 2 hours. Ethyl ether was added to precipitate PEGproduct. The solid was filtered and washed with additional ethyl etherto yield compound 16 (4.4 g, 94%, FIG. 9). The structure was confirmedby ¹³C NMR (67.8 MHz, CDCl₃) with peaks at δ 173.50, 172.50, 155.83,153.01, 150.22, 134. 20, 128.80, 120.55, 69.0-72.5 (PEG), 49.85, 41.16,40.57, 35.91, 29.43, 28.84, 28.66, 26.17, 25.04, 24.35, 24.47, 21.52.

[0249] (Step 3) To a solution of compound 16 (3.3 g, 0.080 mmol),doxorubicin hydrochloride (280 mg, 0.480 mmol), NMM (130 uL, 1.18 mmol),and HOBT (74 mg, 0.480 mmol) in 90 mL of anhydrous DMF and methylenechloride (1:1) was added EDC (120 mg, 0.640 mmol), and the mixturestirred at room temperature overnight. The solvent was evaporated underreduced pressure and the residual solid was recrystallized from IPA (100mL) to give pure compound 17 (2.90 g, 85%).

[0250] The structure was confirmed by ¹³C NMR (67.8 MHz, CDCl₃) withpeaks at δ 213.30, 186.44, 186.16, 172.77, 171.23, 160.55, 155.75,155.13, 153.04, 150.26, 135.37, 134.91, 134.22, 133.38, 133.19, 128.81,127.38, 120.58, 120.38, 119.27, 118.14, 110.97, 110.79, 100.24,69.0-72.5 (PEG), 68.26, 67.27, 67.01, 65.10, 56.21, 51.11, 45.08, 41.02,40.60, 35.91, 35.23, 33.39, 29.43, 28.90, 28.69, 26.22, 25.05, 24.23,22.58, 21.61, 16.48.

Example 8 Confirmation of Efficacy of Tetrapartate Prodrug Relative toDoxorubicin

[0251] The efficacy of PEG-leu-doxorubicin analogs against asubcutaneous human ovarian carcinoma (A2780) injected into nude mice wasdetermined as follows.

[0252] Following at least one week of acclimation, tumors wereestablished in nude mice by injecting 1×10⁶ (100,000) harvested A2780ovarian carcinoma cells in a single subcutaneous site, on the leftaxillary flank region of each animal. The tumor injection site wasobserved twice weekly and measured once palpable. The tumor volume foreach mouse was determined by measuring two dimensions with calipers andcalculated using the formula: tumor volume=(length×width²)/2). Whentumors reached the average volume of approximately 80 mm³, the mice weredivided into their respective experimental groups, which consists ofvehicle (20 m M sodium phosphate in 0.6% NaCl) controls,Leu-Doxorubicin, compound 1 of Example 1, compound 10 of Example 4, andcompound 12 of Example 5). The mice were sorted to evenly distributetumor size, grouped into 6 mice/cage, and ear punched for permanentidentification. Drugs were dosed intravenously via the tail vein onceper week for three weeks (Qd7×3). Mouse weight and tumor sizes weremeasured at the beginning of study and twice weekly through week 5.

[0253] The overall growth of tumors was calculated as the mean tumorvolume at one week following the end of the treatment. A percenttreatment over control (T/C) value was also calculated when the controlgroup's median tumor size reached approximately 800-1100 mm³ and againat one week following treatment. The T/C value in percent is anon-quantitative indication of anti-tumor effectiveness.

[0254] Data is presented in Table 2, below. TABLE 2 PEG-Leu-DoxorubicinTreatment of a Human Ovarian Carcinoma (A2780) Xenograft in NudeMice^(α) Treatment Tumor Volume T/C (%)^(β) Schedule (mean ± sem) At1000 T/C (%)^(β) Compound (mg/kg/dose) Day 21 mm³ At Day 21 Control 04238 ± 356  — — Leu-Dox Qd7 × 3 (30) 659 ± 173 26.3 19.7  Compound 2 Qd7× 3 (30) 619 ± 231 13.2 7.4 Compound 10 Qd7 × 3 (30) 507 ± 173 13.0 8.8Compound 12 Qd7 × 3 (30) 379 ± 134 14.3 6.1

[0255] α- Intravenous treatment in nude mice bearing established tumors(˜80 mm³). n=6/group.

[0256] β- The median tumor volume of treatment and control groups weremeasured and compared when the control group's median tumor volumereached approximately 1000 mm³ and one week after final dosage (day 21).T/C <42% at 1000 mm³ is considered significant anti-tumor activity bythe Drug Evaluation Branch of the NCI.

[0257] Thus, as can be appreciated from the data presented by Table 2,above, the PEG conjugate forms of leu-doxorubicin were more effectivethan the non-conjugated parent compound.

Example 9 Synthesis of Compound 19

[0258] (Step 1) TFA.Alanine-Camptothecin 18 was prepared as described incommonly assigned U.S. Pat. No. 6,127,355, Example 21, the contents ofwhich are incorporated herein by reference.

[0259] (Step 2) 18 (185 mg, 0.36 mmol), was added to solution containing9 (4 g, 0.0982 mmol) in 50 mL anhydrous methylene chloride and DMAP (49mg, 0.40 mmol). The mixture was stirred at room temperature overnight.The reaction mixture was evaporated to dryness and the residual solidrecrystallized two times from 2-propanol (80 mL) to give pure compound19 (3.5 g, 86%). ¹³C NMR (67.8 MHz, CDCl₃) δ 170.80, 167.30, 166.08,156.33, 151.53, 147.99, 146.72, 145.53, 142.07, 133.55, 130.46, 129.61,129.27, 128.89, 127.94, 127.44, 127.02, 119.09, 107.16, 95.35,69.74-65.23 (PEG), 59.13, 49.17, 39.32, 30.76, 21.02, 16.45, 15.36,6.69.

Example 10 Synthesis of Compound 20

[0260] TFA.Alanine-Camptothecin (185 mg, 0.36 mmol) 18 was added to asolution of 11 (4.0 g, 0.0983 mmol) in 50 mL anhydrous methylenechloride and DMAP (49 mg, 0.40 mmol). The mixture was stirred at roomtemperature overnight, the reaction mixture evaporated to dryness, andthe residual solid was recrystallized from 2-propanol two times (80 mL)to give pure 20 (3.5 g, 86%). ¹³C NMR (67.8 MHz, CDCl₃) δ 170.83,166.23, 156.50, 155.21, 152.10, 151.39, 148.20, 145.70, 144.62, 130.75,130.00, 129.76, 129.04, 128.25, 128.03, 127.54, 127.19, 119.45, 107.70,95.44, 69.91-65.25 (PEG), 49.31, 44.90, 30.98, 16.82, 15.31, 15.20,6.81.

Example 11 Synthesis of Compound 21

[0261] TFA.Alanine-Camptothecin (102 mg, 0.179 mmol) 18 and DMAP (37 mg,0.30 mmol) were added to a solution of 5 (2.0 g, 0.0493 mmol) in 40 mLanhydrous methylene chloride. The mixture was stirred at roomtemperature overnight, the reaction mixture evaporated to dryness, andthe residual solid was recrystallized twice from 2-propanol (60 mL) togive pure 21(1.77 g, 87%). ¹³C NMR (67.8 MHz, CDCl₃) δ 171.00, 166.00,161.07, 156.09, 154.00, 151.00, 148.00, 145.50, 135.75, 130.55, 129.56,128.81, 127.75, 127.41, 127.02, 126.69, 120.70, 119.00, 107.70,72.69-67.00 (PEG), 50.70, 48.38, 40.06, 36.86, 30.32, 16.20, 6.65.

Example 12 Synthesis of Compound 22

[0262] A solution of compound 4 (5 g, 0.123 mmol) in toluene (75 mL) isazeotroped with the removal of 25 mL of distillate. The reaction mixtureis cooled to 30° C., followed by the addition of oxalyl chloride (0.031g, 0.246 mmol) and one drop of dimethylformamide. This mixture isstirred for 3 hours at 30-40° C., followed by the addition of2-mercaptothizoline (0.044 g, 0.369 mmol). The reaction mixture isrefluxed for 1 hour, followed by filtration and removal of the solventin vacuo. The crude residue is recrystallized from IPA (100 mL) to yieldcompound 22 (4 g, 90%). The structure is comfirmed by 13C NMR.

Example 13 Synthesis of Compound 23

[0263] Native bovine hemoglobin (bHb) in 100 mM sodium phosphate (pH8.4)/65 mM NaCl buffer is modified to form the conjugated compound 23 asfollows. In a polypropylene container, 20 mL of 22 (0.8 g dissolved in20 mM sodium phosphate/65 mM NaCl buffer at 4° C.) is added to 20 mL ofbHb at 4° C. (22.2 g at 11 mg/mL) with gentle stirring. The pH of thereaction is monitored. The mixture is stirred for 1 hour, the reactionis quenched by the addition of glycine, and stirring is continued for anadditional 15 minutes. Cysteine (dissolved in 100 mM sodium phosphate/65mM NaCl buffer at 4° C., 30 mM final concentration) is added to reduceoxidized hemoglobin (met-Hb) formation, and the reaction mixture isstirred for 16 hours at 4° C. The PEG-Hb is diluted and diafiltered intoformulation buffer (5 mM sodium bicarbonate, 4 mM Na₂HPO₄, 1 mM NaH2PO₄,150 mM NaCl, pH 7.4) to remove the unreacted PEG and/or PEG-glycineconjugate, followed by concentration to 60 mg/mL of compound 23. Thepurity of the PEG-Hb is determined by size exclusion HPLC.

We claim:
 1. A compound of Formula I:

wherein: L₁ is a bifunctional linking moiety; D is a moiety that is aleaving group, or a residue of a compound to be delivered into a cell; Zis covalently linked to [D]_(y), wherein Z is selected from the groupconsisting of: a moiety that is actively transported into a target cell,a hydrophobic moiety, and combinations thereof, Y₁, Y₂, Y₃ and Y₄ areeach independently O, S, or NR₁₂; R₁₁ is a mono- or divalent polymerresidue; R₁, R₄, R₉, R₁₀ and R₁₂ are independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls and substitutedC₁₋₆ heteroalkyls; R₂, R₃, R₅ and R₆ are independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyls, C₁₋₆ alkoxy, phenoxy, C₁₋₈heteroalkyls, C₁₋₈ heteroalkoxy, substituted C₁₋₆ alkyls, C₃₋₈cycloalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, halo-, nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyls andC₁₋₆ alkylcarbonyls; Ar is a moiety which when included in Formula (I)forms a multi-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group; (m), (r), (s), (t), and (u) are independently zeroor one; (p) is zero or a positive integer; and (y) is 1 or
 2. 2. Thecompound of claim 1 , wherein L₁ is selected from the group consistingof:

wherein: M is X or Q; where X is an electron withdrawing group; Q is amoiety containing a free electron pair positioned three to six atomsfrom

(a) and (n) are independently zero or a positive integer; (b) is zero orone; (g) is a positive integer; (q) is three or four; R₇, R₈, R₁₄ R₁₅and R₁₈ are independently selected from the group which defines R₉; andY₅ and Y₆ are independently O, S, or NR₁₂.
 3. The compound of claim 1wherein when y is 2, each of the two D moieties is the same ordifferent.
 4. The compound of claim 1 wherein Z is selected from thegroup consisting of an amino acid residue, a sugar residue, a fatty acidresidue, a peptide residue, a C₆₋₁₈ alkyl, a substituted aryl, aheteroaryl, —C(═O), —C(═S), and —C(═NR₁₆), wherein R₁₆ is selected fromthe same group as R₁₂.
 5. The compound of claim 4 wherein the amino acidresidue is selected from the group consisting of alanine, valine,leucine, isoleucine, glycine, serine, threonine, methionine, cysteine,phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid,lysine, arginine, histidine and proline.
 6. The compound of claim 4wherein the peptide ranges in size from about 2 to about 10 amino acidresidues.
 7. The compound of claim 6 wherein the peptide isGly-Phe-Leu-Gly or Gly-Phe-Leu.
 8. The compound of claim 1 wherein eachD moiety is independently a residue of an active biological material, orH.
 9. The compound of claim 1 wherein each D moiety is independently aresidue of an anticancer agent, an anticancer prodrug, a detectable tag,and combinations thereof.
 10. The compound of claim 9 wherein theanticancer agent or anticancer prodrug comprises an anthracyclinecompound or a topoisomerase I inhibitor.
 11. The compound of claim 9wherein the anticancer agent or anticancer prodrug is selected from thegroup consisting of daunorubicin, doxorubicin, p-aminoaniline mustard,melphalan, cytosine arabinoside, gemcitabine, and combinations thereof.12. The compound of claim 1 wherein at least one D moiety is a leavinggroup selected from the group consisting of as N-hydroxybenzotriazolyl,halogen, N-hydroxy-phthal-imidyl, p-nitrophenoxy, imidazolyl,N-hydroxysuccinimidyl, thiazolidinyl thione, and combinations thereof.13. The compound of claim 1 wherein Ar is selected from the groupconsisting of,

wherein J is selected from the group consisting of O, S, and N—R₁₉, Eand Z are independently C—R₁₉ or N—R₁₉ and R₁₉ is selected from thegroup consisting of hydrogen, C₁₋₆ alkyl, C₃₋₁₂ branched alkyl, C₃₋₈cycloalkyl, C₁₋₆ substituted alkyl, C₃₋₈ substituted cycloalkyl, aryls,substituted aryl, aralkyl, C₁₋₆ heteroalkyl, and substituted C₁₋₆heteroalkyls.
 14. The compound of claim 1 , wherein

comprises an amino acid residue.
 15. The compound of claim 14 , whereinsaid amino acid residue is selected from the group consisting ofnaturally occurring and non-naturally occurring amino acid residues. 16.The compound of claim 1 , wherein (p) is one.
 17. The compound of claim2 , wherein X is selected from the group consisting of O, NR₁₂,

S, SO and SO₂ where R₁₇ is independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₃₋₁₂ branched alkyl, C₃₋₈cycloalkyl, C₁₋₆ substituted alkyl, C₃₋₈ substituted cycloalkyl, aryl,substituted aryl, aralkyl, C₁₋₆ heteroalkyl, substituted C₁₋₆heteroalkyl.
 18. The compound of claim 17 , wherein X is selected fromthe group consisting of O and NR₁₂.
 19. The compound of claim 2 ,wherein Q is selected from the group consisting of C₂₋₄ alkyls,cycloalkyls, aryls, and aralkyl groups substituted with a member of thegroup consisting of NH, O, S, —CH₂—C(O)—N(H)—, and ortho-substitutedphenyls.
 20. The compound of claim 2 , wherein (n) is 1 or
 2. 21. Thecompound of claim 1 , wherein (m) is
 0. 22. The compound of claim 1 ,wherein Y₁, Y₂, Y₃ and Y₄ are O.
 23. The compound of claim 1 , whereinR₁₁ comprises a polyalkylene oxide residue.
 24. The compound of claim 23, wherein said polyalkylene oxide residue comprises polyethylene glycol.25. The compound of claim 1 wherein said polymer residue has a numberaverage molecular weight of from about 2,000 to about 100,000 daltons.26. The compound of claim 1 , wherein said polymer residue has a numberaverage molecular weight of from about 20,000 to about 40,000 daltons.27. The compound of claim 13 , wherein Ar is

wherein r and t are both 1, and R₂ and R₆ are independently H or methyl.28. The compound of claim 1 that is selected from the group consistingof:


29. The compound of claim 28 wherein the polyethylene glycol (PEG) has anumber average molecular weight of from about 20,000 to about 40,000daltons.
 30. A composition comprising a pharmaceutically ordiagnostically effective amount of the compound of claim 1 , where D isa residue of a compound to be delivered into a cell, together with acarrier acceptable for in vivo administration to an animal in needthereof.
 31. A method of preparing a tetrapartate prodrug comprisingreacting a compound of formula:

with a compound of formula: Lx—Z—[D]_(y);  IV wherein B is a leavinggroup for Formula III L₁ is a bifunctional linking moiety; D is a moietythat is a leaving group, or a residue of a compound to be delivered intoa cell; Z is covalently linked to [D]_(y), wherein Z is selected fromthe group consisting of: a moiety that is actively transported into atarget cell, a hydrophobic moiety, and combinations thereof, R₁, R₄, R₉,R₁₀ and R₁₂ are independently selected from the group consisting ofhydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₃₋₈ substituted cycloalkyls, aryls, substitutedaryls, aralkyls, C₁₋₆ heteroalkyls and substituted C₁₋₆ heteroalkyls;R₂, R₃, R₅ and R₆ are independently selected from the group consistingof hydrogen, C₁₋₆ alkyls, C₁₋₆ alkoxy, phenoxy, C₁₋₈ heteroalkyls, C₁₋₈heteroalkoxy, substituted C₁₋₆ alkyls, C₃₋₈ cycloalkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, halo-,nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyls and C₁₋₆ alkylcarbonyls;Ar is a moiety which when included in Formula (III) forms amulti-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group; (m), (r), (s), (t), and (u) are independently zeroor one; (p) is zero or a positive integer; (y) is one or two; and Y₁,Y₂, Y₃ and Y₄ are each independently O, S, or NR₁₂; and R₁₁ is amonovalent or divalent polymer residue.
 32. A method of preparing atetrapartate prodrug comprising reacting a compound of formula

with at least one biologically active material; wherein L₁ is abifunctional linking moiety; La is a leaving group for Formula V; Z iscovalently linked to at least one biologically active material, whereinZ is selected from the group consisting of: a moiety that is activelytransported into a target cell, a hydrophobic moiety, and combinationsthereof; R₁, R₄, R₉, R₁₀ and R₁₂ are independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls and substitutedC₁₋₆ heteroalkyls; R₂, R₃, R₅ and R₆ are independently selected from thegroup consisting of hydrogen, C₁₋₆ alkyls, C₁₋₆ alkoxy, phenoxy, C₁₋₈heteroalkyls, C₁₋₈ heteroalkoxy, substituted C₁₋₆ alkyls, C₃₋₈cycloalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, halo-, nitro- and cyano-, carboxy-, C₁₋₆ carboxyalkyls andC₁₋₆ alkylcarbonyls; Ar is a moiety which when included in Formula (V)forms a multi-substituted aromatic hydrocarbon or a multi-substitutedheterocyclic group; (m), (r), (s), (t), and (u) are independently zeroor one; (p) is zero or a positive integer; Y₁, Y₂, Y₃ and Y₄ are eachindependently O, S, or NR₁₂; and R₁₁ is a monovalent or divalent polymerresidue.
 33. A method of treating a disease or disorder in an animal,that comprises administering a pharmaceutically acceptable compositioncomprising an effective amount of a compound of claim 1 , where D is amoiety that is a leaving group, or a residue of a compound to bedelivered into a cell; to an animal in need thereof.
 34. A method ofdelivering a biologically active material D into a cell in need oftreatment therewith, comprising the process of administering a compoundof claim 1 to an animal comprising said cell, wherein Formula I ishydrolyzed in vivo extracellularly to yield:

wherein Y* is the remainder of Y₂, and is independently selected fromthe group consisting of HO—, HS—, or HNR₁₂—;

and CO₂, and a compound of Formula I-(iii) Z—[D]_(y) is released;wherein Y** is the remainder of Y*, and is independently selected fromthe group consisting of O, S, or NR₁₂; and Z—[D]_(y) crosses themembrane of the cell, and is hydrolyzed therein to release D.